The overall objectives of this proposal are to delineate the structural characteristics of the insulin-sensitive adipocyte hexose transport system and to reveal the molecular basis of insulin action on this system. Experiments completed over the previous project period have led to the partial purification of the adipocyte hexose transporter using classical molecular sieve and ion exchange chromatography to resolve detergent-solubilized membrane proteins. A reconstitution procedure we developed has been used to assay the resolved fractions for transport activity. We propose to pursue three major thrusts to achieve our goals: First, we will continue efforts to purify the insulin-sensitive hexose transporter by standard chromatographic methodology with one key change in our approach--the use of nonionic detergent octylglucoside rather than cholate to facilitate the effective use of ion exchange chromatography to obtain high resolution. Second, we will employ a method to reconstitute the adipocyte transporter into small vesicles which will be made to contain only 1 (or none) protein per vesicle. This theoretically allows quantitation of the number of transporters in membranes from control vs. insulin-treated cells as well as a potentially powerful purification technique. Such purification should be achieved by separation of vesicles (prepared in high glucose solutions) containing the transporter from those not containing the transporter on density qradients as discussed in detail in this proposal. Finally, attempts to raise polyspecific and monospecific antibodies (hybridoma cells) against the adipocyte hexose transporter will be made. Successful preparation of these reagents should allow identification and purification of the transport system.